Cd326 apc

Pancreatic tissue isolation was performed as previously^{36 (link)} with minor modifications. Briefly, pancreas was minced into small pieces, and transferred to 3 mL 0.5 mg/mL Collagenase P (Roche, 11213873001) dissolved in HBSS together with 5 mM glucose. Then the digestion medium was put into the water bath at 37 °C 5 min for acclimatization and gently shaking another 5 min for digestion. To stop the digestion, 10 mL DMEM containing 1% FBS was added to the medium. Tissues were pipetted up and down for further dissociation and filtered by the 70-μm filter. Then cells were centrifuged for 5 min at 1200 rpm and incubated with 1 mL red blood cell lysis buffer (eBioscience, 00-4333-57) at room temperature for 5 min. 10 mL cold PBS containing 1% FBS was added to stop digestion, followed by centrifugation. 1% Fc in PBS was added for 5 min to block, then the isolated cells were stained with fluorochrome-conjugated antibodies including CD45-APC (eBioscience, 17-0451-82, 1:400), CD31-APC (eBioscience, 17-0311-82, 1:40), CD140a-APC (eBioscience, 17-1401-81, 1:100), CD326-APC (eBioscience, 17-5791-82, 1:200) for 30 min at 4 °C. After washing with cold PBS, cells were resuspended in PBS containing DAPI (1:1000). Then flow cytometry experiments were performed using the Beckman Cytoflex LX or sorted using the Sony MA900 flow cytometer.